DESCRIPTION: The Notch protein is a cell surface receptor that plays a key role in numerous phases of development and differentiation. Notch participates in cell-to-cell signaling, becoming activated upon binding to its ligands which are transmembrane proteins on adjacent cells. Defects in Notch signaling cause numerous developmental deformities in organisms from Drosophila to mammals, including human diseases such as T cell lymphomas, a type of cerebral arteriopathy (CADASIL), and Alagille syndrome. We have recently shown that Notch is modified with two unusual forms of 0-linked glycosylation, 0-fucose and 0-glucose, on the epidermal growth factor-like (EGF) modules in its extracellular domain. Over half of Notch's 36 tandem EGF modules contain putative consensus sequences for the addition of these sugars, and most of these sites are evolutionary conserved. Even more significantly, we have very recently discovered a biological role for the 0-fucose modifications by showing that the Fringe protein, a known modulator of Notch function, is an 0-fucose specific 131,3 N-acetylglucosaminyltransferase. These results strongly suggest that Fringe mediates its affects on Notch function by altering the 0-fucose structures on Notch. The modulation of Notch signaling by elongation of 0-fucose provides a new paradigm for the involvement of glycosylation in signal transduction. Our hypothesis is that alterations in the 0-linked carbohydrate modifications on the EGF modules of Notch play a key role in regulation of Notch Signaling. Our data demonstrating Fringe functions through alterations in O-fucose structures strongly supports this hypothesis, and the studies described here will explore it further. In Aim 1, using standard biochemical and molecular biological approaches, we will map the actual sites of 0-fucose glycosylation on Notch. In particular, we will focus our efforts on identification of the 0-fucose sites affected by Fringe. In Aim 2, we will eliminate these sites by generation of point mutations and determine which of them are required for Fringe to function. Then, we will utilize these mutations to analyze how elongation of 0-fucose on Notch results in a change in Notch signaling. In Aim 3, we will continue to identify and characterize enzymes responsible for addition of sugars to 0-fucose on EGF modules. Based on our studies with Fringe, any enzyme which modifies 0-fucose could potentially regulate Notch function. Since many proteins are predicted to be modified with 0-fucose, these enzymes may be involved in regulation of signaling events in other contexts as well.